title: T14(aCGH)

description: T14(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

source_name: no metastases, COPD

tag: Sample code

tag: Age at time of surgery:	72

tag: Packyears:			53

tag: Tumor type

tag: Location tumor:		Left upper lobe

tag: Metastases:			no

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			84

tag: COPD:				COPD

tag: FEV1/FVC:			65 %

tag: GOLD stage:			GOLD II


title: T09(aCGH)

status: Public on Oct 01 2008

type: genomic

city: Groningen

first: Mirjam

last: Boelens

address: Hanzeplein 1

country: Netherlands

institute: UMCG

description: T09(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

hyb_protocol: AMOUNT OF HYBRIDISATION-MIX Take note of the area of the array and establish the size of the lifterslip and the amount of necessary (pre-)hybridisation mix: Lifterslip	Volume hybridisation-mix 22 x 22 mm	30 µl 22 x 30 mm	50 µl 22 x 60 mm	100 µl This protocol is intended for 50 µl hybridisation mix. If necessary take in to account that the amount of labelled product should be increased or decreased. PREHYBRIDISATION - Block the array slide according to the protocol of the manufacturer. - Prepare the prehybridisation-mix by adding together (per 50 µl prehybrydisation-mix): 50 µl salmon sperm DNA (~10g/l), 2 µl 3M NaAc pH5.3, 100 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. at 4 oC in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate - Apply the necessary amount of prehybridisation-mix to the array, on an heated block and place a fresh ethanol cleaned lifterslip on top of the array, without introducing air-bubbles. - Prehybridise for 2 hours in a waterbath at 65oC using a hybridisation chamber. - Wash off the lifterslip with milliQ-water, and spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. PREPARING THE HYBRIDISATION-MIX (For 50 µl of hybridisation mix) - Add together: 200 µl COT1-DNA (1µg/µl), 10 µl 3M NaAc pH5.3, 470 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate. Perform next steps as much in the dark as possible! LABELING The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen). - Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water - Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler. Briefly centrifuge and place on ice for 10 min. - Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase - Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC. - Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns. - Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube. - Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.) - Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min. - Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour. HYBRIDISATION - Denature the DNA in the hybridisation mixture at 100 oC for 5 min. - Apply the suitable amount of the denatured mix to the array, on a heated block; place a fresh, ethanol cleaned, lifterslip on top of the array without introducing air bubbles. - Hybridise for ~40 hours in the Gemini Twin shaker waterbath at 65oC using a hybridisation chamber. POST HYBRIDISATION STEPS - Transfer the slide to a washing rack - Wash the slides using the washing solutions described in the Schott Nexterion guide: 10 min. washing solution 1 at 65oC, 5 min. washing solution 2 at 20oC, 5 min. washing solution 3 at 20oC - Spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. - Scan the array using the Agilent scanner with optimal settings according to the manual. INTERNAL CONTROL Use a reference-DNA sample with opposite gender for these hybridisations. Check the altered ratio’s after a calculation procedure. On the array’s control spots are present, derived from Total Human DNA, CoT-DNA and Drosophila DNA. The intensities of these spots were taken into account in the calculation procedure.

scan_protocol: Agilent scanner (resolution 5 um) in combination with Agilent software. Default settings and protocols as supplied by Agilent

data_processing:
The scanned images were processed with Bluefuse v3.5 (BlueGnomeLtd, Cambridge, UK). A block-median normalization excluding controls was applied to the 2log-transformed Cy3/Cy5 ratios. Subsequent exclusion criteria were: confidence <0.3, quality <1 and SD of replicates >0.5. Log-ratios of replicates were combined using the fusion algorithm. The resulting log-ratios were imported into Nexus-software Copy Number-V3.1 (BioDiscovery, US) to visualise and analyse copy number changes within or between subsets of SCC.
The confidence flag filter selection: A and B (= confidence > 0.7)
Postprocessing protocol:
- Normalization then exclusion
- Block median normalization excluding controls
Next, exclude spots if:
- ID = controls, THEN
- Confidence < 0.3, THEN
- Quality < 1, THEN
- Replicates with StdDEV > 0.5
Next:
- Replicates are combined based on “Name”, using “Fusion” algorithm
Remark: Although this software package includes algorithms that deal with the (local) background, the background values are not listed in any of the output files.

molecule: genomic DNA

organism: Homo sapiens

extract_protocol:
LASER DISSECTION MICROSCOPY
- Frozen tumor sections of 8μm were used for laser dissection microscopy (LDM)
- Only vital tumor cells without apparent admixture of inflammatory cells through the tumor fields were isolated using laser dissection microscopy
- An area of approximately 25x106 μm2 was microdissected from the section using P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany).
- Laser microdissected cells were collected in 20 μl SE buffer
DNA ISOLATION USING A STANDARD SALT-CHLOROFORM EXTRACTION PROTOCOL:
- Add 400 μl SE buffer, 20 μl 10% SDS and 2 μl Proteinase K (20 mg/ml)
- Incubate the solution overnight at 55°C in the incubator; also incubate the saturated NaCl solution (6M) overnight at 55°C
- Add 134 μl preheated saturated NaCl solution to the solution
- Add 1 volume (556 μl) chloroform
- Mix 1 hour on a top-over-top rotor
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Transfer upper phase to a clean tube
- Add 1 volume isopropanol and mix carefully
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Wash pellet/cloud with 200 μl 80% ethanol
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Pipette off all the ethanol, shortly centrifuge and pipette off again
- Dry pellet on air for 5-10 minutes
- Dissolve pellet in 20 μl TE-4
- The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE)
For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY):
AMPLIFYING THE GENOMIC DNA
In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega).
1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly.
2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice.
3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour.
4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature.
PURIFYING THE AMPLIFIED DNA
1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate.
2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate
3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube.
4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g).
5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield.
6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage.
INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION
The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used.
DNA Quality	Total Yield (ug)
No input	<0.5
Poor		<1
Intermediate	1-3
Good		3-10
Excellent	>10
The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform.
LABELING FOR ARRAY ASSAYS
1. Direct Labeling of Unamplified DNA
- 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results.
- >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg).
- Poor input DNA should not labeled.
2. Labeling of Amplified DNA
- 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis.
- 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA.
Only samples scored for Excellent or Good were used for further processing.

tag: Tissue type

value: Human lung tumor (frozen)

tag: Gender

value: Male

tag: Age at time of surgery:	83

tag: Packyears:		45

value: Squamous cell lung carcinoma (SCC)

tag: Type of Surgery:		Lobectomy

tag: TNM stage:		T2N0M0

tag: Tumor stage:		IB

tag: Metastases:		no

tag: Time to distant metastases as from surgery (months):	not applicable

tag: Location of distant metastasis:			not applicable

tag: Dead/Alive:					alive

tag: Survival as from SCC resection (months):		37

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I

source_name: Reference sample

label_protocol:
Prior to the hybridisation, the genomic "test" and "reference" DNA are labelled with Cy3 and Cy5 fluorescent dyes respectively. The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

extract_protocol:
Whole blood was collected in disodium EDTA containers and stored at -20ºC and a number of samples were also stored at -80ºC. To facilitate haemolysis of RBCs it is recommended to store a fresh sample for a few hours in a freezer as freezing destroys the red cells. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added. The solution is left for 10 minutes at RT with occasional mixing by inversion followed by centrifugation for 5 minutes at 4000 rpm. After centrifugation the supernatant is discarded and a white pellet will be observed at the bottom of the tube. This pellet must be washed for at least 3 times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer in order to clean the white blood cells from remaining heme. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing 100mg/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are added to the pellet. The tubes are then incubated at a temperature of 37-55ºC (optimal temp for Proteinase K activity) overnight in a water bath or incubator. During this step the white blood cells' membranes are denatured and DNA goes out in solution. After the incubation, 1.5mls of SE buffer together with 750ml of 6M (saturated) NaCl are added to each tube, followed by the addition of 3.75mls chloroform. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional mixing for at least 30min. Alternatively you can leave the tubes on a rotator for 1 hour. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal breaking force. After centrifugation 2 phases are observed and care must be taken not to disturb the interphase. During this step DNA is extracted into the supernatant and proteins separated into the lower phase. The upper aqueous phase (containing the DNA) is transferred into a clean and sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the addition of an equal volume of isopropanol. DNA will be precipitated by gentle swirling of the tube and is observed visually as a white thread like strand. Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1ml of 75% ethanol. The DNA is then washed by inversion to clean it from any remaining salts and the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded taking care not to discard the pellet. Repeat this step once more. After discarding the supernatant the pellet is dried from excess ethanol either by using a vacuum centrifuge or by leaving the tubes open and inverted in an oven at around 50 - 65ºC for an hour. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0) and left overnight on a rotator. DNA concentration is determined either by agarose gel electrophoresis or spectrophotometry and adjusted to the desired concentration by adding more TE buffer. It is important to note that before adjusting and reading DNA concentrations one must obtain a homogeneous sample of DNA which is not quite easy acquired since DNA is very viscous. To adjust to lower concentration one must used other quantitation methods such as Picogreen (Molecular Probes) using spectrofluorometry as spectrophotometry is not accurate and sensitive at very low concentrations.
For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY):
AMPLIFYING THE GENOMIC DNA
In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega).
1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly.
2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice.
3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour.
4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature.
PURIFYING THE AMPLIFIED DNA
1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate.
2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate
3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube.
4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g).
5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield.
6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage.
INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION
The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used.
DNA Quality	Total Yield (ug)
No input	<0.5
Poor		<1
Intermediate	1-3
Good		3-10
Excellent	>10
The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform.
LABELING FOR ARRAY ASSAYS
1. Direct Labeling of Unamplified DNA
- 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results.
- >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg).
- Poor input DNA should not labeled.
2. Labeling of Amplified DNA
- 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis.
- 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA.
Only samples scored for Excellent or Good were used for further processing.

tag: Mix of DNA from 20 healthy women


title: T10(aCGH)

description: T10(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

label_protocol:
The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

tag: Sample code

tag: Age at time of surgery:	74

tag: Packyears:		53

tag: Tumor type

tag: Location tumor:		Right lower lobe

tag: TNM stage:		T1N0M0

tag: Tumor stage:		IA

tag: Dead/Alive:					dead

tag: Survival as from SCC resection (months):		116

tag: COPD:				COPD

tag: FEV1/FVC:			69 %


title: T12(aCGH)

description: T12(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	73

tag: Packyears:			35

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			no

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			92

tag: FEV1/FVC:			68 %


title: T16(aCGH)

description: T16(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	74

tag: Packyears:			39

tag: Tumor type

tag: Type of Surgery:		Bilobectomy

tag: Location tumor:		Right upper lobe

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			74

tag: FEV1/FVC:			49 %

tag: GOLD stage:			GOLD II

tag: Mix of DNA from 20 healthy women


title: T13(aCGH)

description: T13(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	51

tag: Packyears:			28

tag: Location tumor:		Right lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			no

tag: Survival as from SCC resection (months):			106

tag: COPD:				COPD

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I


title: T15(aCGH)

description: T15(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	47

tag: Packyears:			30

tag: Tumor type

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left lower lobe

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			71

tag: FEV1/FVC:			53 %


title: T06(aCGH)

description: T06(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

source_name: no metastases, no COPD

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	63

tag: Packyears:		47

tag: Type of Surgery:		Pneumectomy

tag: TNM stage:		T2N0M0

tag: Tumor stage:		IB

tag: Metastases:		no

tag: Time to distant metastases as from surgery (months):	not applicable

tag: Location of distant metastasis:			not applicable

tag: Dead/Alive:					alive

tag: Survival as from SCC resection (months):		60

tag: COPD:				no COPD

tag: FEV1/FVC:			72 %

tag: GOLD stage:			not applicable

tag: Mix of DNA from 20 healthy women


title: T07(aCGH)

description: T07(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	61

tag: Packyears:		25

tag: Tumor type

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Right lower lobe

tag: Survival as from SCC resection (months):		104

tag: FEV1/FVC:			78 %


title: T08(aCGH)

description: T08(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	54

tag: Packyears:		35

tag: Type of Surgery:		Wedge resection

tag: Location tumor:		Left upper lobe

tag: TNM stage:		T10M0

tag: Tumor stage:		IA

tag: Metastases:		no

tag: Survival as from SCC resection (months):		97

tag: FEV1/FVC:			71 %


title: T11(aCGH)

description: T11(aCGH): squamous cell lung carcinoma, no metastases, COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	76

tag: Packyears:			10

tag: Type of Surgery:		Pneumectomy

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			no

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			105

tag: COPD:				COPD

tag: FEV1/FVC:			54 %

tag: GOLD stage:			GOLD I

tag: Mix of DNA from 20 healthy women


title: T03(aCGH)

description: T03(aCGH): squamous cell lung carcinoma, no metastasis, no COPD versus reference sample

source_name: no metastasis, no COPD

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	53

tag: Packyears:		40

tag: Tumor type

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left lower lobe

tag: TNM stage:		T2N0M0

tag: Tumor stage:		IB

tag: Metastases:		no

tag: Time to distant metastases as from surgery (months):	not applicable

tag: Location of distant metastasis:			not applicable

tag: Dead/Alive:					alive

tag: Survival as from SCC resection (months):		173

tag: COPD:				no COPD

tag: GOLD stage:			not applicable


title: Brain_22A-Scrapie_End-point_PR06-11_Cy5_Top

status: Public on Sep 30 2008

type: other

city: Winnipeg

first: Reuben

last: Saba

state: Manitoba

address: 1015 Arligton Street

department: Medical Microbiology

country: Canada

institute: University of Manitoba: Public Health agency of Canada

description: no additional information

hyb_protocol: Tagged-cDNA hybridization followed the protocol outlined in the 900 miRNA RT Kit. A hybridization mixture consisting of the differentially tagged cDNA (10 μL of Cy3-labelled and 10 μL of Cy5-labelled targets) and 2 × SDS-based Hybridization Buffer pre-heated to 70°C (20 μL) was mixed and incubated at 75–80°C for 10 minutes, cooled to 50°C until loading and added to the microarray; specifically a 22 × 40 mm cover slip (mSeries Lifterslip™) (Erie Scientific) was centered over the grids and the preheated hybridization mixture was loaded under the cover slip. Microarrays were incubated overnight (16–20 hours) at 50°C in a dark humidified chamber (Genetix). Following hybridisation the cover-slips were removed and the arrays were washed in 2 × SSC, 0.2% SDS wash buffer preheated to 42°C for 15 minutes, 2 × SSC wash buffer at room temperature for 10–15 minutes, and 0.2 × SSC wash buffer at room temperature for 10–15 minutes. Arrays were dried by centrifugation at 1000 rpm for 2–3 minutes and the 3DNA system containing the fluorescent cyanine molecules were hybridized to the arrays; in this case the hybridization mixture contained Cy3 3DNA Capture Reagent (2.5 μL), Cy5 3DNA Capture Reagent (2.5 μL), Nuclease Free Water (15 μL), and 2 × SDS-based Hybridization Buffer. The mix was heated to 70°C for 10 minutes, cooled to 62–64°C and hybridized to the arrays for 4 hours at 62–64°C in a dark humidified chamber. Finally, the arrays were washed as previously described.

data_processing: Microarrays were scanned using Agilent G2565AA and Agilent G2565BA Microarray Scanner System (Agilent). Feature extraction was performed using Array-Pro™ analyzer version 4.5 (Media Cybernetics). Data were stored and normalized in the GeneTraffic Microarray Database and Analysis System (Iobion Informatics, La Jolla, CA). The raw data was filtered so that individual spots had to pass a number of quality criteria including, minimum intensity levels, minimum signal to background ratios, and no greater than 10% missing values for each gene. Genes who passed these criteria were used for further data analysis. Each slide was then normalized using a linear regression, smoothing algorithm (Loess best-fit) over individual array sub grids. Log2 ratios were used for further statistical analysis.

molecule: other

organism: Mus musculus

source_name: PBS treated (IC) VM mouse (age matched) whole brain.

label_protocol: Array 900 miRNA RT Kit (Genisphere) was used to prepare labelled cDNA targets for microarray hybridization according to the manufacturer's protocol. Briefly, 250 ng of LMW enriched RNA was used as a template in a 100 μl Poly(A) Tailing and RT reaction containing 1× Reaction Mix, 2.5 mM MnCl2, 1 mM ATP, and 4 μL poly A polymerase (E-PAP Enzyme). After incubation at 37°C for 15 minutes the reaction was placed on ice and 2 μL of Cy3 or Cy5 reverse transcriptase primer was added. The reaction mix was incubated at 65°C for 10 minutes prior to addition of 23 μL of a second master mix containing per reaction: 10 μL of 5× First Strand Buffer, 5 μL of 0.1 M DTT, 2.5 μL of dNTP Mix, 1 μL of Superase-in RNase Inhibitor, 2 μL of SuperScript II Reverse Transcriptase (200 U) (Invitrogen), and 2.5 μL of nuclease-free water. Subsequently, the reaction was incubated at 42°C for one hour. Finally, 8.75 μL of 0.5 M NaOH/50 mM EDTA and 65°C for 15 minutes was used to inactivate Superscript II. Samples were concentrated to a volume of approximately 10–15 μL, using Microcon YM-10 Centrifugal Filter Devices (Fisher Scientific) according to the manufacturer's protocol.

extract_protocol: Low molecular weight (LMW) RNA enrichment from whole mouse brain was performed using mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer's protocol. LMW RNA was DNase-treated for microarray target preparation using TURBO DNA-free™ (Ambion).

tag: PBS treated (intracranial; IC) VM mouse (age matched) whole brain at end point (>200 dpi).

source_name: 22A scrapie infected (IC) VM mouse whole brain.

tag: 22A scrapie infected (intracranial; IC) VM mouse whole brain at end point (>200 dpi).


title: Brain_22A-Scrapie_End-point_PR06-13_Cy3_Top

description: no additional information

scan_protocol: Microarrays were scanned using Agilent G2565AA and Agilent G2565BA Microarray Scanner System (Agilent). Feature extraction was performed using Array-Pro™ analyzer version 4.5 (Media Cybernetics).


title: Brain_22A-Scrapie_End-point_PR06-13_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-11_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-11_Cy3_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-11_Cy3_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-14_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-13_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-13_Cy5_Top

description: no additional information


title: T02(aCGH)

description: T02(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

extract_protocol:
DNA ISOLATION USING A STANDARD SALT-CHLOROFORM EXTRACTION PROTOCOL:
- Add 400 μl SE buffer, 20 μl 10% SDS and 2 μl Proteinase K (20 mg/ml)
- Incubate the solution overnight at 55°C in the incubator; also incubate the saturated NaCl solution (6M) overnight at 55°C
- Add 134 μl preheated saturated NaCl solution to the solution
- Add 1 volume (556 μl) chloroform
- Mix 1 hour on a top-over-top rotor
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Transfer upper phase to a clean tube
- Add 1 volume isopropanol and mix carefully
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Wash pellet/cloud with 200 μl 80% ethanol
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Pipette off all the ethanol, shortly centrifuge and pipette off again
- Dry pellet on air for 5-10 minutes
- Dissolve pellet in 20 μl TE-4
- The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE)

tag: Sample code

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	67

tag: Packyears:		25

tag: Location tumor:		Left upper lobe

tag: Survival as from SCC resection (months):		151

tag: FEV1/FVC:			71 %

label_protocol:
Prior to the hybridisation, the genomic "test" and "reference" DNA are labelled with Cy3 and Cy5 fluorescent dyes respectively. The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 250 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

extract_protocol: Whole blood was collected in disodium EDTA containers and stored at -20ºC and a number of samples were also stored at -80ºC. To facilitate haemolysis of RBCs it is recommended to store a fresh sample for a few hours in a freezer as freezing destroys the red cells. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added. The solution is left for 10 minutes at RT with occasional mixing by inversion followed by centrifugation for 5 minutes at 4000 rpm. After centrifugation the supernatant is discarded and a white pellet will be observed at the bottom of the tube. This pellet must be washed for at least 3 times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer in order to clean the white blood cells from remaining heme. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing 100mg/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are added to the pellet. The tubes are then incubated at a temperature of 37-55ºC (optimal temp for Proteinase K activity) overnight in a water bath or incubator. During this step the white blood cells' membranes are denatured and DNA goes out in solution. After the incubation, 1.5mls of SE buffer together with 750ml of 6M (saturated) NaCl are added to each tube, followed by the addition of 3.75mls chloroform. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional mixing for at least 30min. Alternatively you can leave the tubes on a rotator for 1 hour. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal breaking force. After centrifugation 2 phases are observed and care must be taken not to disturb the interphase. During this step DNA is extracted into the supernatant and proteins separated into the lower phase. The upper aqueous phase (containing the DNA) is transferred into a clean and sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the addition of an equal volume of isopropanol. DNA will be precipitated by gentle swirling of the tube and is observed visually as a white thread like strand. Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1ml of 75% ethanol. The DNA is then washed by inversion to clean it from any remaining salts and the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded taking care not to discard the pellet. Repeat this step once more. After discarding the supernatant the pellet is dried from excess ethanol either by using a vacuum centrifuge or by leaving the tubes open and inverted in an oven at around 50 - 65ºC for an hour. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0) and left overnight on a rotator. DNA concentration is determined either by agarose gel electrophoresis or spectrophotometry and adjusted to the desired concentration by adding more TE buffer. It is important to note that before adjusting and reading DNA concentrations one must obtain a homogeneous sample of DNA which is not quite easy acquired since DNA is very viscous. To adjust to lower concentration one must used other quantitation methods such as Picogreen (Molecular Probes) using spectrofluorometry as spectrophotometry is not accurate and sensitive at very low concentrations.

tag: Mix of DNA from 20 healthy women


title: T01(aCGH)

description: T01(aCGH): squamous cell lung carcinoma, lymph node metastasis, no COPD versus reference sample

source_name: lymph node metastasis, no COPD

tag: Packyears:		30

tag: Tumor type

tag: Type of Surgery:		Lobectomy

tag: TNM stage:		T2N1M0

tag: Tumor stage:		IIB

tag: Metastases:		lymph node

tag: Time to distant metastases as from surgery (months):	not applicable

tag: Survival as from SCC resection (months):		80

tag: FEV1/FVC:			80 %


title: Brain_22A-Scrapie_End-point_PR06-14_Cy5_Top

description: no additional information

tag: PBS treated (intracranial; IC) VM mouse (age matched) whole brain at end point (>200 dpi).

tag: 22A scrapie infected (intracranial; IC) VM mouse whole brain at end point (>200 dpi).


title: Brain_22A-Scrapie_End-point_PR06-11_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-14_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-12_Cy3_Bottom

description: no additional information


title: T04(aCGH)

description: T04(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	76

tag: Packyears:		55

tag: TNM stage:		T2N0M0

tag: Tumor stage:		IB

tag: Metastases:		no

tag: Location of distant metastasis:			not applicable

tag: Dead/Alive:					dead

tag: Survival as from SCC resection (months):		71

tag: COPD:				no COPD

tag: FEV1/FVC:			70 %

tag: GOLD stage:			not applicable


title: Brain_22A-Scrapie_End-point_PR04-46_Cy3_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-46_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-12_Cy5_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-46_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-41_Cy5_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-46_Cy5_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-12_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-41_Cy5_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR06-14_Cy3_Top

description: no additional information


title: T05(aCGH)

description: T05(aCGH): squamous cell lung carcinoma, no metastases, no COPD versus reference sample

tag: Age at time of surgery:	59

tag: Packyears:		6

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Left upper lobe

tag: Time to distant metastases as from surgery (months):	not applicable

tag: Dead/Alive:					alive

tag: Survival as from SCC resection (months):		64

tag: FEV1/FVC:			77 %

tag: Mix of DNA from 20 healthy women


title: Brain_22A-Scrapie_End-point_PR06-12_Cy3_Top

description: no additional information

tag: 22A scrapie infected (intracranial; IC) VM mouse whole brain at end point (>200 dpi).

tag: PBS treated (intracranial; IC) VM mouse (age matched) whole brain at end point (>200 dpi).


title: Brain_22A-Scrapie_End-point_PR04-41_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-39_Cy3_Bottom

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-39_Cy3_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-39_Cy5_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-41_Cy3_Top

description: no additional information


title: Brain_22A-Scrapie_End-point_PR04-39_Cy5_Bottom

description: no additional information


title: Red_III

status: Public on Sep 21 2008

type: RNA

city: Rishon Le-Zion

first: Igor

middle: Michael

last: Kolotilin

address: Dereh Ha-Macabim

department: Plant sciences

country: Israel

institute: Volcani inst.

description: Biological replicate 3 of 3 (for Red stage)

hyb_protocol: Hybridization of the labeled targets to the tomato microarray slide TOM1 as a probe, was carried out at 42°C for 14 to 16 h using a covered hybridization oven (Amersham Pharmacia, Buckinghamshire, England). Microarray slides were washed seven times for 10 min each: at room temperature in 2XSSC/0.1% SDS- twice, room temperature in 0.2XSSC/0.1% SDS- twice, room temperature with 0.2XSSC- twice, and once in 0.2XSSC at 42°C. Excess fluids were drained from the arrays by centrifugation (5 min at 450g).

scan_protocol: Microarray-derived data acquisition was as in Kolotilin et al., 2007, yet, with several adjustments: 1. Different Agilent DNA microarray scanner (Agilent Technologies) was utilized for microarray scan and image generation; 2. Spotreader (Niles Scientific, CA, USA) software was used for visual-to-numeric data transformation 3. GeneSpring GX (Agilent Technologies) was used to facilitate the analysis of the row numeric data; 4. Low-quality data were identified and discarded when signal-to-noise ratio was <1.5.

data_processing: GeneSpring (GeneSpring GX; Silicon Genetics, Redwood City, CA) was used for Lowess normalization, where 40% of the data served for smoothing. Channel1 (control channel) values lower than 10 were set to 10. Channel2 was divided by Channel1, and this ratio was Log2 transformed.

molecule: polyA RNA

organism: Solanum lycopersicum

source_name: red fruit pericarp

label_protocol: Three microgram of amplified mRNA products were subjected to reverse transcription and then labeled with Cy3 and Cy5 (Amersham Biosciences, Uppsala, Sweden) by the indirect amino-allyl method (Invitrogen, Carlsbad, CA, USA).

growth_protocol: Representative plants of the transgenic line 579HO, homozygous for ySAMdc transgen, and their  isogenic azygous counterparts were planted in a randomized-block design (3 blocks, 5 plants in each block) in an environmentally controlled greenhouse at the Volcani Center, Bet Dagan, Israel, during the winter seasons of 2004 and 2005.

extract_protocol: Total RNA was isolated from ~1g of pericarp tissue of mature-green, breaker and pink-red fruits harvested from the transgenic ySAMdc-overexpressing plants and their azygous isogenic counterparts using Qiagen RNeasy Midi Kit (Qiagen Inc., Valencia, CA, USA). RNA isolation was according to the manufacturer's recommendations with the following modifications: 1. tissue was pulverized in RLT buffer containing β-mercaptoethanol, and 2. RNA samples were treated, on-column for 1 hour, with DNase I using Qiagen RNase-free DNase Set.

treatment_protocol: No treatments were applied

tag: azygous control

tag: transgenic ySAMdc


title: CD14monocytes_LPS+rapamycin_vs._LPS_4h_donor4

city: Vienna

first: Thomas

last: Weichhart

address: Waehringerstraße 10

department: Center for Pathobiochemistry and Genetics

country: Austria

institute: Medical University of Vienna

description: Comparison of the transcriptional effects of rapamycin treatment on human LPS-activated CD14 monocytes.

hyb_protocol: The fluorescent labelled probes were subsequently hybridized on topic-defined PIQORTM Immunology Microarray Human Antisense and subjected to overnight hybridization using a hybridization station.

data_processing: Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGeneâ software (Biodiscovery Inc., El Segundo, USA). Low-quality spots were flagged and excluded for data analysis. Unflagged spots were analysed using the PIQORTM Analyzer software (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The PIQORTM Analyzer allows automated data processing of the raw data text files derived from the ImaGeneâ software. This includes background substraction to obtain the net signal intensity, data normalization and calculation of the Cy5/Cy3 ratios. As an additional quality filtering only those spots/genes are taken into account for the calculation of the Cy5/Cy3 ratio for which at least in one channel the signal intensity > 2-fold than the mean background.

molecule: total RNA

label_protocol: Probe labelling was performed according to the PIQOR Instruction Manual.

extract_protocol: Cell pellets were shipped to Miltenyi Biotec on dry ice. RNA was isolated according to standard extraction protocols (Trizol, Sigma-Aldrich). The following samples were quality checked via the Agilent 2100 Bioanalyzer system. For the linear amplification 1μg of each total RNA was used. Amplified RNAs (aRNAs) were subsequently checked with the Bioanalyzer 2100 system.

treatment_protocol: 10^7 CD14 monocytes were incubated with medium for 90 minutes and then 100ng/ml LPS was added for 4 hours.

treatment_protocol: 10^7 CD14 monocytes were incubated with 100nM rapamycin for 90 minutes and then 100ng/ml LPS was added for 4 hours.

tag: 10^7 CD14+ isolated monocytes from buffy coat.


title: Red_I

description: Biological replicate 1 of 3 (for Red stage)


title: CD14monocytes_LPS+rapamycin_vs._LPS_4h_donor1

tag: CD14+ isolated monocytes from buffy coat.


title: Breaker_IV

description: Technical replicate Dye Swap (for Breaker stage)

source_name: breaker fruit pericarp


title: Red_II

description: Biological replicate 2 of 3 (for Red stage)


title: Red_IV

description: Technical replicate Dye Swap (for Red stage)


title: Mature-Green_II

description: Biological replicate 2 of 3 (for Mature Green stage)

source_name: mature-green fruit pericarp


title: Mature-Green_IV

description: Technical replicate Dye Swap (for Mature Green stage)


title: CD14monocytes_LPS+rapamycin_vs._LPS_4h_donor2


title: CD14monocytes_LPS+rapamycin_vs._LPS_4h_donor3


title: Mature-Green_I

description: Biological replicate 1 of 3 (for Mature Green stage)


title: Breaker_III

description: Biological replicate 3 of 3 (for Breaker stage)


title: Breaker_I

description: Biological replicate 1 of 3 (for Breaker stage)


title: Breaker_II

description: Biological replicate 2 of 3 (for Breaker stage)


title: Mature-Green_III

description: Biological replicate 3 of 3 (for Mature Green stage)


title: Genetic interactions between a set2 null mutation and histone H3/H4 alleles

status: Public on Sep 19 2008

city: Baltimore

first: Daniel

last: Yuan

state: MD

address: 733 N. Broadway, Suite 351

department: Molecular Biology and Genetics

country: USA

institute: Johns Hopkins Univ School of Medicine

description: Survey of genetic interactions between a set2 null mutation and histone alleles.

hyb_protocol: Labeled extracts were hybridized overnight to microarray slides at 42 degrees C. See PubMed ID 15994458.

scan_protocol: Images were acquired with a GenePix 4000B scanner.

data_processing: Paired image files were analyzed with the GenePix Pro software package (MDS Analytical Technologies, version 6.1). Datafiles were subjected to spatial normalization using the hoptag R package (Pubmed 17060357). Using ad hoc R functions (available on request), feature data for each histone mutant were extracted, reduced to their medians, and corrected for a baseline signal intensity derived from the non-histone features in the array. Because the assumptions underlying loess normalization could not be justified, systematic intensity differences between the Red and Green microarray channels were corrected by subtracting estimates based on the estimated mode of the strongest signals. These corrections were checked using scatterplots of the intensity data for the two channels. Data in which neither channel exceeded 2.0 log2 units over baseline were regarded as missing. UpTag and DnTag data were averaged if both values were available and within 2.0 log2 units of each other; otherwise, the larger value was used.

organism: Saccharomyces cerevisiae

source_name: Cells selected for the set2 query mutation

label_protocol: UpTag and DnTag sequences were amplified from genomic DNA by asymmetric PCR using labeled primers. See PubMed ID 15994458.

growth_protocol: Cells were obtained as a pool of haploid mutants, each of which carried a null deletion allele of the hht1-hhf1 locus and a validated replacement of the hht2-hhf2 locus by a synthetic HHT2-HHF2 allele. Growth media and conditions are described separately for each experiment.

extract_protocol: Genomic DNA was prepared by extraction with phenol-chloroform and ethanol-precipitated. See PubMed ID 15525520.

tag: Histone mutants

value: Pooled viable H3 or H4 mutants

tag: Query mutation

tag: Growth medium

value: YPD +G418 (+Nourseothricin to select against mistargeting to hht1-hhf1::natMX).

source_name: Cells selected for the ho query mutation


title: TS32.17 T cell line compared to 3B11 B cells 2nd replicate

status: Public on Sep 20 2008

city: Silver Spring

first: Sai

middle: P

last: Majji

address: 503 Robert Grant Avenue

department: Malaria

institute: Naval Medical Research Center

description: Total RNA was extracted and following reverse transcription, cDNA was synthesized with 3DNA capture sequences at the 5' end. The cDNA was hybridized to the microarray and hybridization was detected following a second round of hybridization using Oyster 550/650 labeled capture reagent (Genisphere).

hyb_protocol: Hybridization following genisphere end labeling method at 50 C using enhanced hybridization buffer.

scan_protocol: Scanned using ScanArray express (Perkin Elmer ver. 2.3).

data_processing: Scanning of the array was performed. Artifacts and irregular hybridization spots were removed by visual inspection and LOWESS normalization was performed with ScanArray express software. Genespring software was used for further data and statistical analysis.

label: Oyster 550

organism: Ictalurus punctatus

source_name: TS32.17 T cell line

label_protocol: Genisphere 3DNA 900 end labeling protocol

extract_protocol: TRIZOL extraction

tag: Cytotoxic T cell line TS32.17

label: Oyster 650

source_name: 3B11 B cell line

extract_protocol: TRIZOL RNA extraction

tag: B cell line 3B11.


title: TS32.15 T cell line compared to 3B11 B cells 3rd replicate

tag: B cell line, 3B11.

source_name: TS32.15 T cell line

tag: Cytotoxic T cell line TS32.15.


title: Chemostat regrowth of over-represented histone alleles, day 10

description: Competition of the top 27 over-represented histone mutants (from day 10 of chemostat growth) against a wild-type control.

source_name: Cells after chemostat growth

value: Synthetic wild-type and 27 over-represented H3 or H4 mutants

value: Yeast Nitrogen Base + glucose restriction (0.08%) + auxotrophy supplements (40mM tryptophan, 50mM methionine, 100mM leucine and 100mM lysine). Growth conditions:Competitive culture in a chemostat at 30 degrees C for the indicated time.

tag: Growth time

value: 10 days

source_name: Cells before chemostat growth

value: 0 days


title: TS32.17 T cell line compared to 3B11 B cells 4th replicate


title: TS32.15 T cell line compared to 3B11 B cells 4th replicate

tag: The cytotoxic T cell line TS32.15


title: Non-homologous end-joining in histone H3/H4 alleles

description: Survey of histone mutants for proficiency at non-homologous end-joining.

source_name: Cells selected for transformation and NHEJ

tag: Transforming DNA

value: HindIII-digested pRS415 (LEU2) plasmid

value: Synthetic Complete -Ura -Leu.

source_name: Cells selected for transformation alone

value: undigested pRS415 (LEU2) plasmid


title: TS32.15 T cell line compared to 3B11 B cells 2nd replicate

tag: The B cell line, 3B11.

tag: The cytotoxic T cell line TS32.15.


title: TS32.17 T cell line compared to 3B11 B cells 1st replicate


title: Genetic interactions between a ubp8 null mutation and histone H3/H4 alleles

description: Survey of genetic interactions between a ubp8 null mutation and histone alleles.

source_name: Cells selected for the ubp8 query mutation


title: TS32.15 T cell line compared to 3B11 B cells 1st replicate


title: Mixed lymphocyte culture compared to 3B11 cells 4th replicate

source_name: Mixed lymphocyte culture

tag: Mixed lymphocyte culture obtained following stimulation of peripheral blood lymphocytes of channel catfish.


title: sgs1 tetrad 16, spore A, B, C, D

status: Public on Sep 16 2008

city: San Francisco

first: Jennifer

last: Fung

state: CA

address: S316 Genentech Hall,  MC2200  600 16th St.

department: Biochemistry and Biophysics

institute: UCSF

description: DNA genotyping data of progeny from a Dsgs1 S96 x YJM789 hybrid strain

hyb_protocol: 50 ug of fragmented DNA was hybridized for 56hs at 42ºC to Affymetrix S98 Yeast Expression arrays.  Gene chips were washed and stained in an Affymetrix Fluidics Station 450.

scan_protocol: Gene chips were scanned using Affymetrix GeneChip Scanner GCS3000

data_processing: The data were analyzed with GCOS 5.4 using Affymetrix default analysis settings and global scaling as normalization method to generate CEL files from DAT files.  Allelescan software combined information from CEL files  from each of the spores of one tetrad to generate a segregation file (_seg.txt) for that tetrad that genotyped the DNA for each spore as arising from either S96 or YJM789 parental origin.

label: biotin

source_name: spore from a 4-spore viable tetrad originating from a Dsgs1 S96 x YJM789 strain

label_protocol: 55 μg of genomic DNA was digested to 50- to 500-bp fragments  with Dnase I and end-labeled with bio-ddATP as previously described (Winzeler et al., 2003).

growth_protocol: Haploid strains were mated and zygotes were picked after 4 hrs and allowed to grow on YPAD plates for < 3 days  before transferring to 2% potassium acetate sporulation plates at 30ºC. Tetrads were dissected after 3-5 days.  Colonies were grown in 550 ml of YPAD for 59 hrs at 30ºC on a 300 rpm shaker.

extract_protocol: Genomic DNA was purified from 500 ml of overnight YPAD culture using a Qiagen genomic-tip 500/G as instructed by the Qiagen genomic DNA handbook with the modification of extending zymolyase and protease K digestion to 5 hour.

tag: genotype


title: Mixed lymphocyte culture compared to 3B11 cells 2nd replicate

tag: Mixed lymphocyte culture from stimulated peripheral blood lymphocytes of channel catfish


title: sgs1 tetrad 13, spore A, B, C, D


title: 42TA macrophage versus 3B11 B cells 3rd replicate

source_name: 42TA macrophage cell line

tag: Macrophage cell line, 42TA.


title: 42TA macrophage versus 3B11 B cells 4th replicate

tag: The B cell line, 3B11

tag: The macrophage cell line, 42TA.


title: Chemostat growth of histone H3/H4 alleles, day 10, replicate 2

description: Survey of histone mutants for relative evolutionary fitness in a chemostat. Day 10 of growth. Replicate 2 of 2.

tag: Histone mutants


title: Detection of histone H4 alleles, replicate 1

description: Test of discrimination of microarray signals (histone H4 alleles) from background. Replicate 1 of 2.

source_name: Cells selected (ineffectively) for the H3 allele

value: Pool of all available H4 mutants, marked by URA3

tag: Histone H3 alleles

value: K9,14,18,23R (and endogenous wild-type)

tag: Growth medium

value: Synthetic Complete plus the indicated supplements. N.B. The intended replacement of the endogenous wild-type H3 allele did not occur, so ch1 and ch2 should be regarded as interchangeable.

tag: Growth selection

value: -Ura

source_name: Cells prepared in parallel without selection

value: endogenous and synthetic wild-type

value: +Ura


title: Mixed lymphocyte culture compared to 3B11 cells 1st replicate

tag: Mixed lymphocyte culture isolated from the peripheral blood leukocytes of channel catfish.


title: sgs1 tetrad 12, spore A, B, C, D


title: 42TA macrophage versus 3B11 B cells 2nd replicate

tag: RNA from a macrophage cell line, 42TA

tag: B cell line, 3B11


title: sgs1 tetrad 11, spore A, B, C, D


title: sgs1 tetrad 10, spore A, B, C, D


title: sgs1 tetrad 3, spore A, B, C, D


title: Chemostat growth of histone H3/H4 alleles, day 10, replicate 1

description: Survey of histone mutants for relative evolutionary fitness in a chemostat. Day 10 of growth. Replicate 1 of 2.

tag: Growth time


title: Detection of histone H4 alleles, replicate 2

description: Test of discrimination of microarray signals (histone H4 alleles) from background. Replicate 2 of 2.

value: del[1-36] (and endogenous wild-type)


title: sgs1 tetrad 8, spore A, B, C, D


title: sgs1 tetrad 7, spore A, B, C, D


title: 42TA macrophage versus 3B11 B cells 1st replicate

tag: The B cell line 3B11.

tag: Macrophage cell line 42TA.


title: sgs1 tetrad 2, spore A, B, C, D


title: sgs1 tetrad 9, spore A, B, C, D


title: ndj1 tetrad 2, spore A, B, C, D

description: DNA genotyping data of progeny from a Dndj1 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dndj1 S96 x YJM789 strain


title: ndj1 tetrad 5, spore A, B, C, D


title: spo16 tetrad 15, spore A, B, C, D

description: DNA genotyping data of progeny from a Dspo16 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dspo16 S96 x YJM789 strain


title: sgs1 tetrad 1, spore A, B, C, D


title: ndj1 tetrad 3, spore A, B, C, D


title: ndj1 tetrad 11, spore A, B, C, D


title: ndj1 tetrad 12, spore A, B, C, D


title: spo16 tetrad 17, spore A, B, C, D


title: ndj1 tetrad 8, spore A, B, C, D


title: spo16 tetrad 18, spore A, B, C, D


title: spo16 tetrad 16, spore A, B, C, D


title: spo16 tetrad 13, spore A, B, C, D


title: ndj1 tetrad 1, spore A, B, C, D


title: spo16 tetrad 1, spore A, B, C, D


title: msh4 tetrad 11, spore A, B, C, D

description: DNA genotyping data of progeny from a Dmsh4 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dmsh4 S96 x YJM789 strain


title: spo16 tetrad 11, spore A, B, C, D


title: spo16 tetrad 12, spore A, B, C, D


title: msh4 tetrad 9, spore A, B, C, D


title: msh4 tetrad 4, spore A, B, C, D


title: msh4 tetrad 6, spore A, B, C, D


title: msh4 tetrad 5, spore A, B, C, D


title: msh4 tetrad 7, spore A, B, C, D


title: msh4 tetrad 8, spore A, B, C, D


title: msh4 tetrad 3, spore A, B, C, D


title: msh4 tetrad 2, spore A, B, C, D


title: TS32.17 T cell line compared to 3B11 B cells 3rd replicate

tag: Cytotoxic T cell line TS32.17

tag: B cell line, 3B11.


title: msh4 tetrad 1, spore A, B, C, D

tag: genotype


title: zip4 tetrad 50, spore A, B, C, D

description: DNA genotyping data of progeny from a Dzip4 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dzip4 S96 x YJM789 strain


title: zip4 tetrad 49, spore A, B, C, D


title: zip4 tetrad 48, spore A, B, C, D


title: zip4 tetrad 53, spore A, B, C, D


title: zip4 tetrad 51, spore A, B, C, D


title: zip4 tetrad 45, spore A, B, C, D


title: zip4 tetrad 54, spore A, B, C, D


title: zip4 tetrad 52, spore A, B, C, D


title: zip4 tetrad 42, spore A, B, C, D


title: zip4 tetrad 43, spore A, B, C, D


title: zip4 tetrad 40, spore A, B, C, D


title: zip4 tetrad 38, spore A, B, C, D


title: zip4 tetrad 36, spore A, B, C, D


title: zip4 tetrad 31, spore A, B, C, D


title: zip4 tetrad 39, spore A, B, C, D


title: msh4 tetrad 10, spore A, B, C, D


title: zip4 tetrad 33, spore A, B, C, D


title: zip4 tetrad 32, spore A, B, C, D


title: zip4 tetrad 37, spore A, B, C, D


title: Chemostat growth of histone H3/H4 alleles, day 20

description: Survey of histone mutants for relative evolutionary fitness in a chemostat. Day 20 of growth. Continuation of chemostat d10 rep1.

value: 20 days


title: zip4 tetrad 22, spore A, B, C, D


title: zip4 tetrad 21, spore A, B, C, D


title: zip4 tetrad 23, spore A, B, C, D


title: zip4 tetrad 27, spore A, B, C, D


title: zip4 tetrad 17, spore A, B, C, D


title: zip4 tetrad 7, spore A, B, C, D


title: zip4 tetrad 11, spore A, B, C, D


title: zip4 tetrad 9, spore A, B, C, D


title: zip4 tetrad 16, spore A, B, C, D


title: zip4 tetrad 10, spore A, B, C, D


title: zip4 tetrad 18, spore A, B, C, D


title: zip4 tetrad 1, spore A, B, C, D


title: zip4 tetrad 2, spore A, B, C, D


title: zip4 tetrad 3, spore A, B, C, D


title: zip4 tetrad 15, spore A, B, C, D


title: zip3 tetrad 9, spore A, B, C, D

description: DNA genotyping data of progeny from a Dzip3 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dzip3 S96 x YJM789 strain


title: zip3 tetrad 12, spore A, B, C, D


title: zip3 tetrad 3, spore A, B, C, D


title: zip3 tetrad 4, spore A, B, C, D


title: zip3 tetrad 5, spore A, B, C, D


title: zip2 tetrad63r, spore A, B, C, D

description: DNA genotyping data of progeny from a Dzip2 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dzip2 S96 x YJM789 strain


title: zip3 tetrad 2, spore A, B, C, D


title: zip2 tetrad61r, spore A, B, C, D


title: zip3 tetrad 1, spore A, B, C, D


title: zip2 tetrad62r, spore A, B, C, D


title: Mixed lymphocyte culture compared to 3B11 cells 3rd replicate

source_name: 3B11 B cell line

tag: The mixed lymphocyte culture obtained following stimulation of peripheral blood lymphocytes of channel catfish.


title: zip2 tetrad60r, spore A, B, C, D


title: zip2 tetrad53r, spore A, B, C, D


title: zip2 tetrad58r, spore A, B, C, D


title: zip2 tetrad44r, spore A, B, C, D


title: zip2 tetrad41r, spore A, B, C, D


title: zip2 tetrad48r, spore A, B, C, D


title: zip2 tetrad40r, spore A, B, C, D


title: zip2 tetrad43r, spore A, B, C, D


title: zip2 tetrad51r, spore A, B, C, D


title: zip2 tetrad39r, spore A, B, C, D


title: zip2 tetrad39, spore A, B, C, D


title: zip3 tetrad 10, spore A, B, C, D


title: zip2 tetrad59r, spore A, B, C, D


title: zip2 tetrad37, spore A, B, C, D


title: zip2 tetrad35, spore A, B, C, D


title: zip2 tetrad32, spore A, B, C, D


title: zip2 tetrad33, spore A, B, C, D


title: zip2 tetrad56r, spore A, B, C, D


title: zip2 tetrad30, spore A, B, C, D


title: zip2 tetrad31, spore A, B, C, D


title: zip2 tetrad38, spore A, B, C, D


title: zip2 tetrad29, spore A, B, C, D


title: zip2 tetrad28, spore A, B, C, D


title: zip2 tetrad27, spore A, B, C, D


title: zip1 tetrad 19, spore A, B, C, D

description: DNA genotyping data of progeny from a Dzip5 S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a Dzip1 S96 x YJM789 strain


title: zip1 tetrad 18, spore A, B, C, D


title: zip1 tetrad 17, spore A, B, C, D


title: zip1 tetrad 23, spore A, B, C, D


title: zip1 tetrad 16, spore A, B, C, D


title: zip1 tetrad 13, spore A, B, C, D


title: zip1 tetrad 15, spore A, B, C, D


title: wild type tetrad 63, spore A, B, C, D

description: DNA genotyping data of progeny from a S96 x YJM789 hybrid strain

source_name: spore from a 4-spore viable tetrad originating from a wild type S96 x YJM789 strain

value: wild type


title: wild type tetrad 64, spore A, B, C, D


title: zip1 tetrad 1, spore A, B, C, D


title: wild type tetrad 58, spore A, B, C, D


title: zip4 tetrad 6, spore A, B, C, D


title: wild type tetrad 36, spore A, B, C, D


title: wild type tetrad 40, spore A, B, C, D


title: wild type tetrad 38, spore A, B, C, D


title: wild type tetrad 55, spore A, B, C, D


title: wild type tetrad 37, spore A, B, C, D


title: wild type tetrad 49, spore A, B, C, D


title: wild type tetrad 46, spore A, B, C, D


title: wild type tetrad 44, spore A, B, C, D


title: wild type tetrad 39, spore A, B, C, D


title: wild type tetrad 54, spore A, B, C, D


title: wild type tetrad 51, spore A, B, C, D


title: wild type tetrad 34, spore A, B, C, D


title: wild type tetrad 33, spore A, B, C, D


title: zip1 tetrad 30, spore A, B, C, D


title: wild type tetrad 31, spore A, B, C, D


title: wild type tetrad 24, spore A, B, C, D


title: wild type tetrad 30, spore A, B, C, D


title: wild type tetrad 11, spore A, B, C, D


title: wild type tetrad 6, spore A, B, C, D


title: wild type tetrad 10, spore A, B, C, D


title: wild type tetrad 5, spore A, B, C, D


title: wild type tetrad 35, spore A, B, C, D


title: wild type tetrad 65, spore A, B, C, D


title: subject 26 Liver Rejector

status: Public on Sep 15 2008

city: Pittsburgh

first: Rakesh

last: Sindhi

state: PA

address: 3705 Fifth Avenue

department: Hillman Center for Pediatric Transplantation

institute: Children’s Hospital of Pittsburgh

description: Exon array data from child <20 yrs

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Exon Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed with Affy Power Tools (APT) using rma-sketch normalization method.

source_name: whole blood

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

tag: subject 26 Liver Rejector


title: subject 24 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 24 Liver Non-rejector


title: subject 25 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 25 Liver Non-rejector


title: subject 18 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 18 Liver Non-rejector


title: subject 13 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 13 Liver Non-rejector


title: subject 22 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 22 Liver Non-rejector


title: subject 20 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 20 Liver Non-rejector


title: subject 23 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 23 Liver Non-rejector


title: subject 10 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 10 Liver Non-rejector


title: subject 15 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 15 Liver Non-rejector


title: subject 19 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 19 Liver Non-rejector


title: subject 12 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 12 Liver Non-rejector


title: subject 16 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 16 Liver Rejector


title: subject 7 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 7 Liver Rejector


title: subject 11 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 11 Liver Rejector


title: subject 21 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 21 Liver Non-rejector


title: subject 4 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 4 Liver Rejector


title: subject 8 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 8 Liver Rejector


title: subject 6 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 6 Liver Non-rejector


title: Xenopus-ICD-FoxJ1-2

city: La Jolla

first: Chris

last: Kintner

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute

description: explants injected with ICD and FoxJ1, replicate 2

hyb_protocol: 10ug of cRNA was hybridized to Xenopus laevis Affymetrix chips for 16hrs at 45C, then washed and stained using the Affymetrix Fluidics Station

scan_protocol: GeneChip Scanner 3000 7G from Affymetrix

data_processing: The data was analyzed with Microarray Suite 5.0 using the default Affymetrix settings.  Statistical analysis was then performed using  Bullfrog Analysis software and pair-wise comparisons.

organism: Xenopus laevis

source_name: explants from embryos injected with ICD and FoxJ1 RNA

label_protocol: Biotynylated cRNA was prepared according to standard Affymetrix Protocols

growth_protocol: Xenopus embryos grown in 0.1X MMR until stage 9/10

extract_protocol: Total RNA was extracted using the Proteinase K method

treatment_protocol: Animal caps were cut, explanted and cultured in Danilchik's + 0.1% BSA from stage 9/10 until harvest at stage 22

tag: injected at 2-cell stage

tag: explanted at stage 9/10

tag: harvested at equivalent of stage 22


title: Xenopus-ICD-FoxJ1-1

description: explants injected with ICD and FoxJ1, replicate 1


title: subject 14 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 14 Liver Non-rejector


title: Xenopus-ICD-2

description: explants injected with ICD, replicate 2

source_name: explants from embryos injected with ICD RNA


title: Xenopus-ICD-1

description: explants injected with ICD, replicate1


title: subject 17 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 17 Liver Rejector


title: Xenopus-ICD-3

description: explants injected with ICD, replicate 3


title: wild type tetrad 29, spore A, B, C, D

tag: genotype


title: subject 9 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 9 Liver Rejector


title: subject 5 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 5 Liver Rejector


title: +D7Smed_d

status: Public on Sep 12 2008

city: Salt Lake City

first: Alejandro

last: Sánchez Alvarado

state: UT

department: Neurobiology and Anatomy

institute: University of Utah School of Medicine

description: Electronic spot quantification was accomplished using ImaGene software from Biodiscovery.

hyb_protocol: The hybridization was carried out overnight at 42 degrees Celcius in a humid chamber

scan_protocol: The hybridized microarray slides were scanned using an Axon GenePix 4000B Scanner at a 10um/pixel resolution.

data_processing: The Cy3(exp)/Cy5(ref) ratio was calculated using Microsoft Excel, the data was mean scale normalized and log 2 transformed using Spotfire Decision Site Software for Functional Genomics (v7.0).

organism: Schmidtea mediterranea

source_name: Intact planaria 7 days after 100Gy (+D7Smed_d)

label_protocol: 1ug of aRNA was used as a template and random nonamers were used to prime first strand cDNA synthesis using Superscript II in the presence of Cy3-dCTP.  The resulting cDNA/RNA hybrid was treated with 0.25 M NaOH to hydrolyze RNA.  The first strand cDNA molecule was then purified using a Qiagen QIAquick PCR purification kit to remove unincorporated fluorescent nucleotides and primers.  The purified labeled sample was subsequently concentrated through speedvac centrifugation and resuspended in Ambion SlideHyb Glass Array Hyb Buffer#1.

extract_protocol: Total RNA was extracted using Trizol reagent (Invitrogen),  DNAse I treated (Qiagen) at room temperature for 15min, and cleaned up to remove any remaining organics or contaminants using Qiagen RNAeasy spin columns. RNA quality was assessed using an Agilent Bioanalyzer and amplified using the Ambion MessageAmp RNA amplication kit. The quality of the resulting amplified RNA (aRNA) was examined using the Agilent Bioanalyzer and quantified using a Nanodrop spectrophotometer.

tag: Pool of 10 planarians 7 days after exposure to 100Gy

label_protocol: 1ug of aRNA was used as a template and random nonamers were used to prime first strand cDNA synthesis using Superscript II in the presence of Cy5-dCTP.  The resulting cDNA/RNA hybrid was treated with 0.25 M NaOH to hydrolyze RNA.  The first strand cDNA molecule was then purified using a Qiagen QIAquick PCR purification kit to remove unincorporated fluorescent nucleotides and primers.  The purified labeled sample was subsequently concentrated through speedvac centrifugation and resuspended in Ambion SlideHyb Glass Array Hyb Buffer#1.

tag: Pool of 10 intact wild type planarians


title: +D1Smed_d

source_name: Intact planaria 24hr after 100Gy (+D1Smed_d)

tag: Pool of 10 planarians 24hrs after exposure to 100Gy


title: +D7Smed_c

source_name: Intact planaria 7 days after 100Gy (+D7Smed_c)


title: +D7Smed_a

source_name: Intact planaria 7 days after 100Gy (+D7Smed_a)


title: +D1Smed_c

source_name: Intact planaria 24hr after 100Gy (+D1Smed_c)


title: subject 2 Liver Non-rejector

description: Exon array data from child <20 yrs

tag: subject 2 Liver Non-rejector


title: +D7Smed_b

source_name: Intact planaria 7 days after 100Gy (+D7Smed_b)


title: WTSmed_a

source_name: Intact wild type planaria (WTSmed_a)


title: +D1Smed_a

source_name: Intact planaria 24hr after 100Gy (+D1Smed_a)

tag: pool of 10 planarians 24hrs after exposure to 100Gy


title: WTSmed_b

source_name: Intact wild type planarian (WTSmed_b)

tag: Pool of 10 intact wild planarians


title: +D1Smed_b

source_name: Intact planaria 24hr after 100Gy (+D1Smed_b)


title: WTSmed_c

source_name: Intact wild type planaria (WTSmed_c)


title: subject 1 Liver Rejector

description: Exon array data from child <20 yrs

tag: subject 1 Liver Rejector


title: ESC wild type (454)

status: Public on Sep 10 2008

first: James

middle: Graham

last: Ruby

address: 1700 4th Street QB3 s404

department: Biochemistry

data_processing: Small RNA sequences 16-27 nt long are provided with the number of reads for each unique sequence.  Small RNA 5p and 3p ends were determined using matches to ligated linker sequences

source_name: wt ES cells

growth_protocol: Mouse embryonic stem cells were grown on gelatin-coated plates in standard, feeder-free conditions, using MEF conditioned media as previously described (Wang et al. 2007). DGCR8 knockout ES cells were previously described (Wang et al. 2007).  Dicer knockout cells were produced by first deriving ES cells  (Blelloch et al. 2006) from Dicer flox/flox mice (kindly provided to us by Michael McManus - (Harfe et al. 2005)). Resulting cells were treated with cre recombinase to loop out the floxed Dicer alleles.  Homozygous loop out was confirmed by genotyping (Harfe et al. 2005).

extract_protocol: Total RNA from wt, dgcr8Δ/Δ and dicer1Δ/Δ was extracted using Trizol (Invitrogen). Small RNA libraries were constructed essentially as previously described (Lau et al. 2001). Small RNAs ranging from 18 to 32 nucleotides were gel purified and ligated to the 3´ adaptor and 5´ adaptor oligonucleotides. Products from the second ligation were purified, reverse transcribed, and amplified using corresponding primers. Sequences of the adaptors and primers are as published (Illumina, 454 Life Sciences). Samples were sequenced on an Illumina 1G Genome Analyzer and 454 Genome Sequencer.

tag: cultured embryonic stem cells


title: ESC dgcr8 (454)

source_name: dgcr8(-) ES cells


title: 82Q_3

status: Public on Sep 01 2008

city: Bethesda

first: Alexandre

last: Kuhn

address: 35 Convent Dr

department: NIA

institute: NIH

description: Infected 4 weeks prior with TRE-Htt-N853-82Q-expressing recombinant lentivirus

hyb_protocol: Affymetrix Eukaryotic Target Hybridization protocol (GeneChip® Expression Analysis Technical Manual 701021 Rev. 5)

scan_protocol: Affymetrix® GeneChip® Scanner 3000 with GCOS software according to manufactuer's protocol (GeneChip® Expression Analysis Technical Manual 701021 Rev. 5)

data_processing: Probe set summarization and normalization was performed by robust multi-array averaging (RMA, including background subtraction, summarization by median polish and quantile normalization) using the R/Bioconductor package affy.

label: Biotin

organism: Rattus norvegicus

source_name: Primary rat striatal neuron cultures, 4 weeks in vitro

label_protocol: Affymetrix GeneChip® IVT Labeling Kit, according to manufacturer's protocol (GeneChip® Expression Analysis Technical Manual 701021 Rev. 5)

extract_protocol: RNeasy mini system (Qiagen, Hilden, Germany)

tag: Sample group

value: 82Q; Tissue:Lentivirus-infected primary neuronal culture from rat striatum


title: Nodules versus incipient prenodules HopT28WHT28d rep 3-dye swap

city: Lisboa

first: Ana

middle: Margarida

last: Fortes

address: Campus da FCUL

department: Plant Biology

country: Portugal

description: Technical replicate 1 of 1. Comparison of  incipient prenodules developed in medium without hormones to  nodules developed in medium with hormones. Both files coming from Versarray and edited for GEPAS are presented

hyb_protocol: The hybridization mix consisted of 30μl of labeled cDNAs, 7,5μg Cot-1 DNA (Invitrogen), 15μg Poly (A+), 7,5μg of salmon sperm DNA, 1.5μl of 50X Denhard´t solution, 7.9μl 20X SSC and 1.5μl 10% SDS (w/v). Mixture was denatured at 97ºC for 2 min, put on ice and then 1μl of 10x DIG blocking (Roche Diagnostic GmbH, Mannheim, Germany) buffer was added. Hybridization was carried out at 65º C in chambers ArrayIt (Telechem International).

scan_protocol: Slides were scanned using VersArray ChipReader® and spot and background intensities quantified using Versarray Analyser software (Bio-Rad).

data_processing: Data files were imported into GEPAS (http://gepas.bioinfo.cipf.es) and log2-transformed prior to normalization (DNMAD). Print-tip Lowess was used for within-slide normalization considering background subtraction. Impute missing values (median). Patterns with less than 70% removed.

organism: Humulus lupulus

source_name: In vitro culture of hop internodes which developed into incipient prenodules, day 28 without hormones

label_protocol: 70  µg of RNA  was used for cDNA synthesis with a RT primer (oligo dT) for labeling with either Cy3 or Cy5 dye molecules (Amersham, Buckinghamshire, UK) and Revert Aid Hminus Reverse Transcriptase (Fermentas)

growth_protocol: Internodes were kept in vitro on MS medium with IAA, BAP and sucrose

extract_protocol: Total RNA was isolated essentially as described by Rerie et al. (1991, Mol Gen Genet) using phenol/chloroform

treatment_protocol: RNA was extracted from internodes and from material at various morphogenic stages (prenodule, nodule and incipient nodules)

tag: Age

value: day 28 without hormones (control)

source_name: In vitro culture of hop internodes which developed into nodules, day 28

value: day 28


title: 82Q_4


title: 82Q_1


title: Prenodule versus internodes HopT0T15d rep 4

description: Biological replicate 3 of 3. Comparison of prenodules to internodes at beginning of culture. Both files coming from Versarray and edited for GEPAS are presented

source_name: In vitro culture of hop internodes which developed into prenodules, day15

value: day 15

source_name: In vitro culture of hop internodes, day 0

value: day 0 ( at beginning of culture)


title: Nodules versus internodes  HopT0T28d rep 2

description: Biological replicate 1 of 3. Comparison of nodules to internodes at beginning of culture. Both files coming from Versarray and edited for GEPAS are presented

source_name: In vitro culture of hop internodes which developed into nodules, day28


title: 18Q_4

description: Infected 4 weeks prior with TRE-Htt-N853-18Q-expressing recombinant lentivirus

value: 18Q; Tissue:Lentivirus-infected primary neuronal culture from rat striatum


title: Nodules versus incipient prenodules HopT28WHT28d rep 1

description: Biological replicate 1 of 3. Comparison of nodules developed in medium with hormones to incipient prenodules developed in medium without hormones. Both files coming from Versarray and edited for GEPAS are presented


title: 82Q_6


title: Nodules versus internodes  HopT0T28d rep 4

description: Biological replicate 3 of 3. Comparison of nodules to internodes at beginning of culture. Both files coming from Versarray and edited for GEPAS are presented


title: subject 3 Liver Rejector

description: Exon array data from child <20 yrs

source_name: whole blood

tag: subject 3 Liver Rejector


title: 18Q_6


title: 18Q_3


title: 18Q_2


title: 82Q_5


title: 18Q_5


title: Nodules versus internodes  HopT0T28d rep 1-dye swap

description: Technical replicate 1 of 1.  Comparison of  internodes at beginning of culture to nodules. Both files coming from Versarray and edited for GEPAS are presented

value: day 0


title: Nodules versus incipient prenodules HopT28WHT28d rep 4

description: Biological replicate 3 of 3. Comparison of nodules developed in medium with hormones to incipient prenodules developed in medium without hormones. Both files coming from Versarray and edited for GEPAS are presented

source_name: In vitro culture of hop internodes which developed into incipient prenodules, day 28


title: Nodules versus incipient prenodules HopT28WHT28d rep 2

description: Biological replicate 2 of 3. Comparison of nodules developed in medium with hormones to incipient prenodules developed in medium without hormones. Both files coming from Versarray and edited for GEPAS are presented


title: Prenodule versus internodes HopT0T15d rep 3-dye swap

description: Technical replicate 1 of 1. Comparison of  internodes at beginning of culture to prenodules. Both files coming from Versarray and edited for GEPAS are presented

source_name: In vitro culture of hop internodes which developed into prenodules, day 15


title: 18Q_1

extract_protocol: RNeasy mini system (Qiagen, Hilden, Germany) followed by ethanol precipitation


title: Prenodule versus internodes HopT0T15d rep 1

description: Biological replicate 1 of 3. Comparison of prenodules to internodes at beginning of culture. Both files coming from Versarray and edited for GEPAS are presented


title: Nodules versus internodes  HopT0T28d rep 3

description: Biological replicate 2 of 3. Comparison of nodules to internodes at beginning of culture. Both files coming from Versarray and edited for GEPAS are presented


title: Skin Papilloma (Tail)_c-Fos-deficient_sample8 Cy3

city: Stanford

last: Haag

state: Santa Clara

address: 265 Campus Drive

department: ISCBRM

institute: Stanford School of Medicine

description:
Tumor model: mice overexpressing Sos in keratinocytes leading to a constitutive activation of the Ras pathway, which results in formation of skin papilloma.
Experiment: papillomas from c-Fos fl/fl mice (harboring active c-Fos) are compared to papillomas from c-Fos Δ/ Δ mice (lacking c-Fos)

hyb_protocol: Purified, dye-labelled cDNA was mixed with 120 μl UltraHyb hybridization buffer (Ambion), agitated for 30–60 min at 60°C, then for 10 min at 70°C on a thermo mixer and subsequently applied to pre-heated (60°C) microarrays mounted in a GeneTAC Hybridization Station (Genomic Solutions, Ann Arbor). Hybridizations were performed for 23 h at 42°C with gentle agitation. Thereafter, the arrays were automatically washed at 36°C with (i) 0.5× SSC, 0.1% (w/v) SDS for 5 min; (ii) 0.05× SSC, 0.1% (w/v) SDS for 3 min; and (iii) 0.05× SSC for 2 min. Flow time was set to 40 s. Immediately after the completion of the final washing step, the arrays were unmounted, immersed in 0.05× SSC, 0.1% (w/v) Tween 20 and dried by centrifugation in 50 ml Falcon tubes (30 s at 500, 1000 and 1500 r.p.m., respectively, followed by a final step of 240 s at 2000 r.p.m.).

scan_protocol: Hybridized microarrays were scanned in a two-color Agilent Scanner G25505B (Agilent, Santa Clara, CA) at 5 μm resolution and automatically adjusting PMT voltages according to manufactures specification.  Recorded images were subsequently analyzed using GenePix Pro 6.0 software.

data_processing: Data tables of raw intensity values were filtered excluding those features that were either user-flagged, exhibited a foreground intensity mean-to-median ration of more than 0.3 fold change difference or having an overall foreground intensity of below 250. For normalization of filtered features, the variance stabilization normalization algorithm (Huber et al., 2002) was applied. The “VALUE”-column contains the Log-ratio in sample over reference orientation for the respective microarray.

source_name: mouse skin papilloma, c-Fos-deficient, SOS+

label_protocol:
For reverse transcription:
RNA/primer-mix: 2 µg total RNA, 500 ng (dT)-T7 primer, total volume 6 µl
; denature RNA/primer 4' at 70°C, chill on ice;

RT-mix: 2 µl M-MuLV RT buffer (Fermentas), 0.5 µl 10 mM dNTP-mix, 0.5 µl T4gp32, 0.5 µl RNase Inhibitor (Promega) , 0.5 µl reverse transcriptase (RevertAid, Fermentas), total volume 4 µl

; add ice cold RT-mix, mix well
; incubate 1h at 50°C with heated lid, inactivate 15' at 65°C, chill on ice;
For second strand synthesis:
SSS-mix: 15 µl 5x 2nd strand buffer (Invitrogen), 1.5 µl 10 mM dNTP-mix, 2.22 µl DNA Pol. I (9 U/µl) (Promega), 0.1 µl RNase H (10 U/µl) (Epicentre), 0.5 µl DNA Ligase (10 U/µl) (USB), 45.68 µl RNase-free water, total volume 65 µl
; add ice cold SSS-mix to RT reaction, mix well
; incubate 2h at 14-16°C in thermal cycler
; add 3.33 µl T4 DNA-Pol. (3 U/µl) (NEB) , mix by flicking
; incubate 15' at 14-16°C in thermal cycler, heat inactivate 10' at 70°C
; add 75 µl phenol/chlorof./isoamylalc. (pH 8), mix vigorously by pipetting
; transfer to prespun (30'' at 16000g) PLG Heavy 0.5 ml (Eppendorf)
; spin 5' at 16000g
; transfer aq. phase to prepared (resuspend, drain by gravity, 2' at 1000g) P-6 MicroSpin column (BioRad)
; spin 4' at 1000g, recover eluate
; transfer eluate to 0.5 ml PCR tube
; add LPA, 3.5 µl 5 M NaCl, 220 µl 100% Ethanol, mix well
; precipitate 60' at -80°C over night
; spin tube 30' at 16000g, 4°C, remove supernatant
; wash pellet with 500 µl 70% ethanol, spin tube 5' at 16000g, remove supernatant
; resuspend dried cDNA-pellet in 10 µl water

For In Vitro transcription:
IVT-mix (Promega): 6 µl RNase free water, 8 µl 5x T7 Transcr. buffer, 3 µl each 100 mM ATP, GTP, CTP, UTP, 4 µl T7 Enzyme-mix, total volume 30 µl
; add IVT-mix to 10 µl cDNA, mix by pipetting
; incubate 12h at 37°C
For aRNA cleanup:
RLT / β-ME – mix: 3.5 µl β-mercapto-ethanol, 76.5 µl water, 350 µl RLT (Qiagen RNeasy), total	volume 430 µl

; add RLT / β-ME-mix to IVT product (aRNA), mix well
; add 250 µl 100% ethanol, apply to RNeasy Mini column (Qiagen)
; spin 15" at 8000g, discard flow-through, transfer column to new 2 ml tube
; wash with 500 µl RPE, spin 15" at 8000g, discard flow-through
; add 30 µl RNase-free water onto membrane, spin 1' at 8000g,repeat eluting step,
; precipitate aRNA with 1 µl LPA, 30 µl 7.5M ammonium-acetate and 227.5 µl 100% ethanol
; wash pellet with 500 µl 70% ethanol, spin tube 5' at 16000g, remove supernatant
; resuspend in RNase-free water ( 200 µg/µl)
For 2nd Round RT:
RNA/primer-mix: 5 µl aRNA (1 µg), 1 µg N6-random-primer, total volume 6 µl

; RT-mix: 2 µl M-MuLV RT buffer (Fermentas), 0.5 µl 10 mM dNTP-mix, 0.5 µl T4gp32, 0.5 µl RNase Inhibitor (Promega) , 0.5 µl reverse transcriptase (RevertAid, Fermentas), total volume 4 µl

; denature RNA/primer-mix 5' at 70°C, snap cool on ice 2', hold 5' at room temperature, mix well
; incubate:
20' at 37°C
20' at 42°C
10' at 50°C
10' at 55°C
15' at 65°C
hold at 37°C
; add 1 µl RNase H-dilution (1:10 in 5x 2nd strand buffer, (Invitrogen))
; incubate 30' at 37°C, 2' at 95°C
For Klenow-labeling (per channel):
Klenow-mix (Invitrogen): 40 µl 2.5X Random Primer Solution, 10 µl 10X dNTP-(low dTTP)-mix*, 3 µl Cy-labeled dUTP (Cy3 or Cy5), 34 µl water, 2 µl Klenow fragment Exo- (40 U/µl), total volume 89 µl

;  add to cDNA (11 µl), mix gently but thoroughly
;  incubate 16h at 37°C
; add 450 µl TE to labeled cDNA, apply to Microcon YM-30 column (Millipore)
; spin 13’ at 16000g, discard flow-through
; wash with 430 µl TE (measure dye incorporation on NanoDrop), add 18.75 µl blocking-mix**,
; spin 13’ at rpm, discard flow-through
; elute labeled cDNA by inverting column into new tube, spin 8’ at 16000g

* 2 mM dATP, 2mM dCTP, 2mM dGTP, 0.5 mM dTTP
**12.5 µl Cot-1 DNA (1 µg/µl), 2.5 µl poly-A RNA (5 µg/µl), 3.75 µl tRNA (10 µg/µl)

growth_protocol:
Animals were housed in specific pathogen free (SPF) and in light, temperature and
humidity controlled conditions. Food and water were available ad libitum.

extract_protocol: Total RNA was isolated from mouse skin using TRIzol (Invitrogen) following manufacturer’s protocol. (http://www.invitrogen.com/content.cfm?pageid=469)

tag: Tissue

value: skin from tail;

tag: Tumor stage

value: papilloma;

tag: Genetic background

value: mixed 129/Ola-C57BL/6-CBA-LVA-B6C3F1

source_name: Universal Mouse Reference RNA (Stratagene)

extract_protocol: Extraction of total RNA was accomplished by (Stratagene). According to manufacturer’s specification an Absolutely RNA Kit, based on a glassfiber method in combination with guanidine thiocyanate buffer was used.

value: Liver, hepatocyte; Testis, leydig cell; Kidney; Mammary gland; Embryo; Lung, alveolar macrophage; Skin, epidermis; Thymus, T lymphocyte; Meyloma, B lymphocyte; Soleuse muscle, myoblast; Embryo, fibroblast


title: 82Q_2


title: Case 1F, patient with TCRVbeta13.1+/CD4+/Nka+/CD8-/+ dim T-LGL lymphocytosis

city: Salamanca

first: Arancha

last: Rodríguez-Caballero

address: Avda. Universidad de Coimbra. Campus Unamuno

country: Spain

institute: Cancer Research Center. University of Salamanca.

description: Freshly isolated monoclonal CD4+ T-LGL from peripheral blood of male patient with TCRVbeta13.1+/CD4+ T-LGL lymphocytosis

hyb_protocol: Following fragmentation, 25 microg of cRNA were hybridized to the HG-U133 Plus 2.0 Array chip (Affymetrix) according to the protocols provided by the manufacturer.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.

data_processing: RNA expression values for each probe set were calculated using the RMA (“Robust Multi-array Average”) algorithm.

source_name: Freshly isolated monoclonal CD4+ T-LGL from human peripheral blood

label_protocol: 100 ng of total RNA from the purified cell fractions was amplified and labeled using the GeneChip two cycle cDNA synthesis kit and the GeneChip IVT labeling kit (Affymetrix Inc., Santa Clara, CA), respectively.

extract_protocol: Total RNA was isolated from magnetic-activated cell sorter (MACS)-freshly purified unstimulated monoclonal CD4+ T-LGL lymphocytes from PB sample (purity of ≥98%). To get pure and highly concentrated RNA, the silica membrane technology NucleoSpin® RNA XS (Macherey-Nagel, Düren, Germany) was used.

tag: Patient identifier

tag: Sex

value: male

tag: Cell purity of ≥ 98%


title: Skin Papilloma (Tail)_c-Fos-deficient_sample9 Cy5